Drosophila TDP-43 RNA-Binding Protein Facilitates Association of Sister Chromatid Cohesion Proteins with Genes, Enhancers and Polycomb Response Elements
Fig 7
TBPH and Lark preferentially bind RNAs from cohesin-binding genes in vitro using known RNA-binding domains.
Diagrams of the TBPH and Lark protein structures with known sequence domains shown at the top: RRM, RNA Recognition Motif; ZnF, Zinc Finger. The smallest fragments tested that bind RNA in vitro are underlined in red. The bar graphs below the protein diagrams show examples of RNA-binding competition experiments with TBPH (left) and Lark (right). Whether or not the RNA is from a cohesin-binding gene or contains UG repeats is indicated (Y = yes, N = no). The sequences of all short RNAs tested from cohesin-binding and non-binding genes are in S1 Table, along with a summary of their abilities to bind TBPH and Lark derived from multiple experiments. As detailed in the text, soluble His6-SUMO fusion proteins were immobilized on NTA-Zn2+ agarose beads and incubated with equimolar mixtures of the indicated short RNAs. RNAs that were retained after washing were quantified by real-time PCR and binding was defined by enrichment relative to the amount of the CG6310-3 control RNA, which does not bind to either protein. All RNA fragments were tested in two independent binding experiments with freshly made fusion proteins, and each RNA was measured twice in each experiment. Error bars are the standard deviations of all measurements in all experiments. Enrichment of 10-fold or greater is defined as specific binding when recorded in S1 Table. The bottom bar graphs show example RNA-binding experiments with the indicated fragments of TBPH and Lark to map the domains that bind RNA. SDS-PAGE characterization of the immobilized protein fragments and the residues contained within each fragment are shown in S10 Fig. For truncated proteins, RNA enrichment greater than or equal to half the enrichment obtained with the full-length protein in the same experiment was defined as binding. With TBPH, the RRM1-containing region is necessary and sufficient for RNA-binding. For Lark, the zinc finger (ZnF) is required in addition to the RRM-containing domain. It is unknown if one or both of the Lark RRM domains is required to bind RNA.